Technical Field
The present disclosure relates to methods, compositions, and kits for performing PCR, including multiplex PCR.
Description of the Related Art
PCR, especially multiplex PCR, is one of the technologies used for target enrichment to selectively sequence specific regions of interest in genome. The primer sequences used in PCR are considered to be of no use for downstream analysis, and are often removed through enzymatic digestion before the sequencing step so that sequencing capacity is not wasted in sequencing the primer bases.
The primer removal step is accomplished by substituting one or two Thymine (T) bases in the primer with Uracil (U) bases. The PCR reaction proceeds as usual, incorporating Adenine (A) bases into the PCR product at positions complementary to the U bases in the primer. After the PCR step, the double stranded DNA product is treated with uracil-N-glycosylase (UNG, also known as uracil-DNA glycosylase (UDG)), which causes degradation of the primer strand until the furthest 3′ U base, resulting in single-stranded overhangs at both ends of the double-stranded DNA molecule. These single-stranded overhangs are then removed using exonuclease. To minimize the waste of sequencing capacity in sequencing the primer bases, a U base is typically placed as close to the 3′ terminus of the primer as possible.